Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MEq~ OD AN D APPARATU S FOR CAPILLARY : ~;
HYDRODYNAMIC FRACTIONATION
.~ield of the Invention
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~his invention relates generally to the
lS separation of submicron`sized particles by hydrodynamic
fractionation. ` -
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~s~Li~tiQn-Q~ Related Art `j'
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Separation by ~low was first proposed on
theoretical grounds by DiMarzio and Guttman (P .lymer.
L~L~ ~ 2~7 (1969)). According to their analysis, r
separation by flow according to particle size is due to
two factors: (i) the radial velocity profile developed ~i~
by a fluid moving through a capillary tube allowing the 2.,
particles to move at di~ferent speeds and (ii) the
inability of larger particles to approach the capillary '.
wall as closely as smaller particles, which causes the j
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larger particles to sample fluid streamlines of higher i~
velocity, movin~3 on the average at speeds greater than
the average eluant velocity. '~
Separation of micron sized particles by
flow through tubes has been described by Noel et al. i;~
Chromat~L~h~ L~h, 373 (1978)) and Mullins and Orr i;~`
(l~t~ ha~ El~ 79 (1979)) using long
capillary tubes (50 to 200 meters in length), with l,;
inner diameters in the range from 250 to 500 micr~ns,
to fractionate particles with diameters greater than
one micron. Although these investigator,s achieved
separations between submicron particles and particles
larger than a micron, they were not able to fractionate
lS mixtures of submicron particles. Brough and coworkers
(~L ~h~ 95~ 2Q~, 175 (1981)) used smaller
capillary tubes (150 microns in diameter) in an effort
to expand the size range of the fractionation.
~lthough Brough and coworkers were able to detect
differences in elution times between submicron
particles, their resolution was not sufficient to 1~-
resolve bimodal mixtures of submicron parti~cles. j~ `
de Jaeger et al. (~1 Charact. ~, 187, (1986)) improved
the resolution of the separation by using a slightly
smaller diameter capillary (100 microns) in conjunction
with a block copolymer, dissolved in the eluant stream,
that absorbs on the capillary wall and the particle
surface. These investigators were able to detect the
presence of submicron particles in polydisperse samples
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containing mixtures of dif~erent monodisperse
standards.
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S~mm~L~ of the Tnvention '~
This invention provides an apparatu8 and
method for the complete fractionation of submicron ;~;~
particles according to size by capillary hydrodynamic
fractionation. This objective is achieved by using '~
small diameter capillaries; introducing a minor l~
fraction of a liquid dispersion of particles to be ;~i
separated into at leas~ one capillary; pa'ssing the
minor fraction through the capillary; and, at the exit
of the capillary, diluting the minor fraction with the
additional solvent. These modifications in the flow '~
patterns are essential to the use of capillaries with
diameters smaller than 60 microns. This invention is
especially adapted for rapid analytical separation of
~ not only rigid colloidal particles but also of soft
latexes, ultrahigh molecular weight biopolymers, and ;,~
macromolecules.
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Fig. 1 is a schematic of the capillary
hydrodynamic fractionator of this invention.
Figs. 2-8 are representative spectrometer
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tracings o~ separated dispersions. ,~
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etailed ne~cription of the Tn~ention '~
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As used herein, the term "monodisperse~ means ,'t,~'
that the dispersed particles are all of essentially one
size and the term "polydisperse~ means that,the
disp~rsed particles include a range of sizes.
1 0 j.
- In accordance with the invention there is ,~
provided an apparatus and method ~or the,separation of
a polydisperse dispersion of particles in a fluid by ~.
size which comprises passing the dispersion of
particles through a capillary tube whose diameter is `~
several times larger than~the particles to, be separated ''i'''```
and eluting t~he capillary tube with a further portion '
o the dispersing medium whereby the larger particles ,~
of the dispersion elute from the capillary ~irst and
successively'smaller particles elute subsequently.
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A schematic diagram of a typical ,~
~ractionation system employing the principles of this ,;
invention is shown in Fig. 1. ReserYoir 10 supplies ,~¢`
solvent, optionally with a surfactant, via pump 12 to
stream splitter 14. Th~ sample of particles dispersed
in liquid is introduced via injection port 16 situated ,r~
upstream from stream splitter 14. A major portion of
the stream is discharged to waste; a minor fraction of ',~'
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the stream passes throu~h one or more capillary
~- hydrodynamic frac~ionation (CHDF) tubes 18 where the ,~
particles are separated by velocity profile. After ~.
passage through CHDF tubes 18, the separated dispersion ~'~is diluted with additional solvent, or make-up fluid, t~pumped via tube 20, at stream merger ~2. The diluted, ,~
`; separated sample is then passed to a suitable analyzer, ;~
such as UV detector 24, opkionally interfaced to
computer 26.
1 0
In one embodiment, a pump, such as a
Laboratory Data Control Model 1396-57 dual pump with a
pulse dampener was used to pump the eluant through the `~
capillary tube. This pump is capable of a maximum
pressure of 5000 psi and the flow rate can be adjusted ~t-from 5~0 ml/h down to ~9 ml/h. The sample is injected ~'~into the eluant stream, without interrupting flow to
the capillary, through a Rheodyne Model 7413 sample ,~'injection valve with selectable sample loops of 0.5, 1,
~ and S 1.
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The open capillary tubes used were of fused
silica and were supplied by Polymicro Technologies, in
lengths o~ 1 to 50 m, with diameters of 4, 7, 14, 34,
~5 and 60 microns.
Since the problems caused by dead volume in
the injection and detection systems are considerably
more severe with these narrow capillaries than with
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l~rger inner diameter capillaries, the flow around both
injection and detection systems has been modified. In
order to minimize dead volume effects, the eluting
solution was split into two streams after passing
through the injection valve, while at the exit o~ the ;~
capillary more eluant was added to the stream entering
the detector cell. The sample splitting and make-up ;~
ratios used ranged from 1:100 to 1:107 and above
depending on the diameter of the capillaries and flow `~
rate through the microcapillary. For every combination ;~
of capillary and ~low rate, the splitting and make-up l~
ratios that give the least peak spreading should be
used. " is~
` A minipump was used to pump the make-up
eluant, which is mixed with the fluid exiting from the ,~
capillary through the detector cell. The detector used ~`
was a Laboratory Data Control SM 4000 Programmable UV-
Li~ht Detector fitted with a 14- 1 flow cell. The ;t~
~ colloid and marker species were detected in the :~
effluent by monitoring turbidity at 220 nm (but other l;~
wavelengths can be used~. The output from the detector t~
was monitored both on a strip chart recorder and also ~1
digitally with a disk drive interfaced to the detector
~5 through an Analog Devices DAS1155 A/D converter and a
Frequency Devices four-pole Bessel active filter.
Digital data analysis was carried out on a l`~
microcomputerr and the processed re~ults were output to
a dot matriY printer.
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The eluant is generally the same solvent ~s
` that in which the sample to be fractionated is ~i"
dispersed. Preferably, the eluant contains one or more
sur~actants, in a concentration o~ from O.OOOlM to
0.1 M such as sodium lauryl sulfate or a
pol~oxyalkylene glycol. Most preferably, the
sur~actant is a mixture of sodium lauryl sulfate and
polyoxyethylene lauryl alcohol.
` A wide variety of capillary tubes may be
~lployed in the present invention. Genexally, the ``~
shape of the capillary is not critical.' ~owever, mOst
conveniently employed is a cylindrical capillary tube. ?
15 The capillàry tube desirably has a surface which is j~
generally inert to the dispersing or suspending medium
employed; that is, it is insoluble in the dis~persing
medium, and if coated by solutions in the dispersing
medi~, it will not absorb particles onto the capillary
20 wall. It is essential and critical in the practice of
the present invention that the particles being
separated do not adhere to the inner wall of the ~ 7
capillary tube and form multiparticle layers thereon. 1'~
The capillary tubes may be constructed of a wide i~
25 variety of materials such as, for example, fused
silica, glass, plastics and metal. Eminently ~ i~
satisfactory for many applications are fused silica
glass or plastic tubes from about 3 to 30 microns '
inside diameter. Advantage usly, the process of the
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present invention is used for separating polydisperse
synthetic latex particles which range in si~e from
about lO0 Angstroms to as large as l to 5 microns, and
is preferably employed in separating polydisperse latex .,~,
particles having a size range of from lO0 Angstroms to
2 microns and most advantageously having a size range
of O.OS to 0.5 microns. Usually it is desired that the~`~
inside diameter of the capillary tube be from 5 to 50 $
times the diameter of the largest latex particles to be
separated. Typically, the capillary tube has a length '~
varying from lO centimeters to 50 or lO0 meters or ,~
more, depending upon the degree oE separation desired."~,
Typical operating pressures for capillaries 7 microns
in diameter and 3 meters in length generally are from `~`
about 30Q to 6000 pounds per square inch. For most ':!~
applications, particularly Eor small scale laboratory ~
operations or for analytical procedures, it is usuallyT'~'
desirable to employ a capillary tube of small diameter~,T~7
arld substantial length, such as 5 microns in diameter`T~_
and lO meters in length. Such capillaries, if formed if
with Elexible silica tubing or synthetic plastic tubing
or Elexible metal tubing, may be conveniently coiled to ;~
occupy a minimum of space. More than one capillary
tube, connected in series or in parallel or both, may
~5 be employed.
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Ea~IPL E
A mixture was prepared of two monodisperse ,'~
~ polystyrene latexes having particle diameters o~ 0.357 '~
i~ 5 micron and 0.109 micron respectively. The polydisperse i~
latex so formed was diluted to 3 weight percent with
deionized water containing 10-3 molar sodium lauryl
sulfate. The capillary tube has an average diameter of
4 microns. Deioni~ed water containing the surfactant
was pumped through the system at a rate of l milliliter ,,~
per minute, of which 1.13 x 10-7 l/min pass through
the capillary. 0.005 milliliters of the latex mixture
` was introduced through the sample injection valve and '~
the turbidi;ty of the effluent recorded on a strip chart
recorder. A distinct separation of the 0.109 micron i
and 0.357 micron particles~was observed after a period
of about 3 minutes. The plot of absorbency versus time
of Fig. 2 indicates the relatively sharp separation of
the larger and smaller particles.
EX~MPLE 2
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The procedure of Example 1 was repe~ated with
a mixture of 0.234 micron and 0.357 micron monodisperse '`
latexes. The plot, as shown in Fig. 3, consists of a
bifurcated peak, with the peak of the larger siæe
particles appearing 155 seconds after sample injection
and the peak of the smaller particles 163 seconds after
injection.
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EX~IPL E 3 1,~
-- The procedure of Example 2 was repeated with '~
- 5 the exception that the average velocity of the eluant ~ t-'
through the capillary tube was reduced to 0.67 x 10-7
l/min. A complete separation of ~he two latexes was l~r;
obtained as set forth in Fig. 4.
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EX~IPL E 4 ~ `
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The procedure of Example 1 was'rep~ated with
a mixture of particles with diameters of 0.357 and 1.1
microns injected into the system, connected to a
capillary tube o~ diameter 34 microns and length 20
meters. The flot~ rates through the injection valve ~'
were 0.41, 0.29, and 0.18 ml/min while thè
corresponding flow rates through the capillaries were
- ~, 3 and 1.8~Ll/min respectively. A partial separation 1~?~;'
of the two latexes was obtained as indicated in Fig 5,
which is a representation of the trace obtained from
the recordinq of the light scattering detector, for j~
different eluant averaqe velocities. Contrary to what i~
~5 was observed with mixtures of submicron particles, ;~
illustrated in Examples ~ and 3, in the case of samples
containing particles with diameter greater than 0.5 ~:
microns, the efficiency of the separation decreases ~.
with increasing eluant velocity (shorter elution time).
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EX~MPL E 5 `.
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A mixture was prepared of polystyrene latex ,;~
particles having diameters of 0.357 micron and 1.1 ~`;,!i`
micron and injected into the system of Example 4. The "~
positive displacement pump delivered 1 milliliter per ~'
minute of an aqueous solution of a non-ionic sur~ace li~
active agent sold under the trade designation of
Pluronic F - 108 (B~S~), in a concentration of 0.15 gram i~
per liter to the capillary. The ~low rate through the
capillary tube was 29 ~ l/min. Good separation o the
latex particles was obtained, as is indicated in i`~
Fi9. 6.
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EX~MPL E 6 , !~`
When the procedure o~ Example 1 was repeated
~mploying a polydisperse latex, a curve was obtained
which is characteristic of the paxticle size
distribution. Fig. 7 illustrates the fractionation !`~
obtained with a mixture of paxticles of OolO9~ 0~176~ ;
0.234, and 0.357 microns in diameter. ''~
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EX~IPL~ 7 i
An aqueous solution of 10-4 molar sodium
lauryl sulfate and 0.1% by weight of polyoxyethylene ~i
lauryl alcohol was pumped through the system at a rate
of 1.3 ml/min. The capillary tube used had an inside ,~
diameter o~ 7.0 microns and a length of 5m. Flow ii
throuqh the capillary was at a rate of 2.5 x 10-5 `~x
ml/min. ~ mixture of polystyrene latex particles
- 10 havinq diameters of 0.109, 0.176, 0.234 and 0.357 '
microns was injected into the flowing aqueous solution !~
of sodium lauryl sulfate and polyoxyethylene lauryl ~;~
alcohol. A complete separation of the ~our latexes was p,
obtained as set forth in Fig. 8. In other experiments, `;
larger particles were bètter separated with a smaller
concentration of polyoxyethylene. ~`~
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